Enhancing inorganic carbon fixation by photosynthetic organisms

ABSTRACT

A method of enhancing inorganic carbon fixation by a photosynthetic organism. The method is effected by transforming cells of the photosynthetic organism with an expressible polynucleotide encoding a polypeptide having a bicarbonate transporter activity. Preferably, the polynucleotide further includes a plant promoter. Sequences and constructs for implementing the method are also described.

This is a continuation of U.S. patent application Ser. No. 09/332,041,filed Jun. 14, 1999. FIELD AND BACKGROUND OF THE INVENTION

[0001] The present invention relates to a method of enhancing inorganiccarbon fixation by photosynthetic organisms, nucleic acid molecules foreffecting the method, and transformed plants characterized by enhancedinorganic carbon fixation.

[0002] Photosynthesis is a process executed by photosynthetic organismsby which, inorganic carbon (Ci), such as CO₂ and HCO₃ ⁻, is incorporatedinto organic compounds using the energy of photon radiation.Photosynthetic organisms, such as, soil grown and aquatic plants andcyanobacteria (blue-green algae), depend on the organic compoundsproduced via photosynthesis for sustenance and growth.

[0003] The rate of photosynthesis is determined by several parameterswhich include but are not limited to, CO₂ concentration, O₂concentration, temperature, light intensity, and the water balance inthe case of soil grown plants.

[0004] Of the above mentioned parameters photosynthesis is largelyinfluenced by the rate with which the carboxylating enzyme ribulose1,5-bisphosphate carboxylase/oxygenase, (rubisco) can fix available CO₂.The rate of CO₂ fixation depends on the concentration of CO₂ and to alesser extent on the concentration of O₂ which are available to rubisco,since CO₂ fixation competes with photorespiration, which is the processof O₂ fixation by rubisco. Furthermore, when photosynthesis israte-limited by the supply of CO₂, the imbalance between the lightenergy input and its dissipation via CO₂ fixation, leads to photodynamicdamage to the photosynthetic reaction centers (photoinhibition).

[0005] Since rubisco posseses a very slow turnover rate, in order tomeet the energy requirements of photosynthetic organisms it needs to bepresent in abundance within the photosynthetic cells thereof(approximately 50% of the soluble protein). The abundance of rubiscoleads to deleterious effects to the energetic balance of photosyntheticcells since most available resources of these cells must be allocated tothe production of rubisco.

[0006] To overcome the problems associated with inefficient CO₂ fixationat low CO₂ concentrations, many photosynthetic organisms have evolvedvarious mechanisms for concentrating the inorganic carbon exposed torubisco. Such mechanisms are found in certain higher plants (belongingto the C4 group, including maize and sorghum) and in aquaticphotosynthetic organisms. In C4 plants, differential expression andtight regulation of several genes enables cooperation between themesophyll and bundle sheath cells leading to elevated CO₂ concentrationin the latter. An initial carboxylation reaction, where HCO₃ ⁻ serves asthe substrate, occurs in the mesophyll cells. The product is thentransferred to the bundle sheath cells where it is decarboxylated(releasing the CO₂ fixed in the mesophyll cells) in close proximity torubisco (confined to these cells). C3 plants, to which most of the cropplants belong, are unable to concentrate CO₂ at the site of rubisco andtherefore grow poorly (compared to C4 plants) under water-limitingconditions. Due to the large number of genes involved, and to thecomplexity of their tight, spatial regulation, in the operation of theC4 mechanism, the introduction of the whole C4 CO₂ concentratingmechanism to C3 plants is presently impossible.

[0007] Many aquatic photosynthetic microorganisms possess induciblemechanisms that concentrate CO₂ at the carboxylation site, compensatingfor the relatively low affinity of rubisco for its substrate, thusallowing acclimation to a wide range of CO₂ concentrations [25]. Thepresence of membrane-located mechanisms for inorganic carbon (Ci)transport are central to these concentrating mechanisms.

[0008] Photosynthetic microorganisms including cyanobacteria are alsocapable of acclimating to a wide range of CO₂ concentrations. Theprocess of acclimation is mediated via changes, effected at variouscellular levels, which include modulation of gene expression involved inthe operation of the CO₂ concentrating mechanism (CCM) [1-5]. Thismechanism enables these photosynthetic microorganisms to raise the CO₂level at the carboxylation site thus overcoming the large (5 to 20-fold)difference between the K_(m)(CO₂) of rubisco and the concentration ofdissolved CO₂ when at equilibrium with the surrounding atmosphere. Incyanobacteria, the components of the CCM include energy-dependent HCO₃ ⁻transport, CO₂ conversion to HCO₃ ⁻ [3] and highly organized structures,termed carboxysomes, where carbonic anhydrase catalyzes the formation ofCO₂ from HCO₃ ⁻ in close proximity to rubisco [1-3]. The 5 activity ofthe CCM increases dramatically following transfer from high to low CO₂concentrations mainly due to changes in the Ci transport capabilitiesand an increase in the number of carboxysomes [3, 6, 7]. Some of thegenes involved in the operation of the CCM were identified with the aidof high-CO₂-requiring mutants but there is little information on thosedirectly involved in HCO₃ ⁻ uptake [3, 4, 8].

[0009] The ability to concentrate CO₂ provides distinct advantages tophotosynthetic organisms. The photosynthetic rate of such CO₂concentrating organisms is not severely affected by lower CO₂concentrations and as a result, the growth and productivity of suchorganisms are not severely limited by the environmental concentration ofCO₂, and by water, in the case of soil grown plants.

[0010] Therefore, it is highly desirable to enhance CO₂ fixation in nonCO₂ concentrating photosynthetic organisms, such as C3 plants, since inall probability, such an enhancement would directly result in improvedgrowth and productivity.

[0011] There are two possible biotechnological approaches with which anenhanced rate of CO₂ fixation can be achieved.

[0012] One such approach involves the manipulation of rubisco by sitedirected mutagenesis in order to raise both its affinity and specificityto CO₂ (compared with O₂) and to further enhance its turnover rate.Although numerous studies were conducted in order to isolate a rubiscomutant which posseses the above mentioned improvements at present nosuch rubisco mutants have been isolated.

[0013] Another approach for enhancing CO₂ fixation involves raising theconcentration of CO₂ outside the cells of higher plants. In higherplants the concentration of CO₂ in the air spaces within the leaves isdetermined by the diffusional flux of CO₂ through the stomata from thesurrounding air via the unstirred layer around the leaves. The stomatalconductance for CO₂ is largely determined by the water balance of theplants. Modulation of stomatal opening by water availability isexercised by plants in order to combat water stress. Stomatal closure,in order to minimize water loss under conditions of water stress,generates a significant resistance to CO₂ diffusion leading to a sharpdecline in CO₂ fixation. Although raising the concentration of CO₂ in aclosed environment, such as a greenhouse, is commonly practiced in orderto raise the diffusional flux of CO₂ and as such, raise plantproductivity, such a practice however, is not applicable to outdoorgrown plants.

[0014] Increasing stomatal conductance can theoretically serve to raiseplant productivity, but at present, viable mechanisms for enhancing thestomatal CO₂ conductivity have not been proposed.

[0015] Thus, at present, both of the above mentioned approaches aretheoretical and as such cannot be applied to render photosyntheticorganisms, such as C3 plants, more efficient at fixing CO₂.

[0016] There is thus a widely recognized need for, and it would behighly advantageous to have, a method of enhancing the CO₂ fixation inphotosynthetic organisms, such as higher plants, especially C3 plants.

SUMMARY OF THE INVENTION

[0017] According to one aspect of the present invention there isprovided a method of enhancing inorganic carbon fixation by aphotosynthetic organism, the method comprising the step of transformingcells of the photosynthetic organism with an expressible polynucleotideencoding a polypeptide having a bicarbonate transporter activity.Preferably, the polynucleotide further includes a plant promoter.

[0018] According to another aspect of the present invention there isprovided a nucleic acid molecule for enhancing inorganic carbon fixationby a photosynthetic organism, the nucleic acid molecule comprising apolynucleotide encoding a polypeptide having a bicarbonate transporteractivity. Preferably, the nucleic acid molecule further comprising aplant promoter being upstream to the polynucleotide effective inexpressing the polypeptide.

[0019] According to yet another aspect of the present invention there isprovided a transformed photosynthetic organism comprising the nucleicacid molecule described herein.

[0020] According to further features in preferred embodiments of theinvention described below, the step of transforming the cells of thephotosynthetic organism with the expressible polynucleotide encoding thebicarbonate transporter is effected by a method selected from the groupconsisting of genetic transformation and transient transformation.

[0021] According to still further features in the described preferredembodiments the genetic transformation is effected by a method selectedfrom the group consisting of Agrobaterium mediated transformation,electroporation and particle bombardment.

[0022] According to still further features in the described preferredembodiments the transient transformation is effected by a methodselected from the group consisting of viral transformation,electroporation and particle bombardment.

[0023] According to still further features in the described preferredembodiments the polynucleotide includes (i) a nucleic acid sequencecorresponding to at least a portion derived from SEQ ID NO:2, theportion encodes the protein having the bicarbonate transporter activity;(ii) a nucleic acid sequence at least 60% identical to the portion, asdetermined using the Blast software where gap penalty equals 10 forexistence and 10 for extension, average match equals 10 and averagemismatch equals −5; (iii) a nucleic acid segment hybridizable with theportion under hybridization conditions of hybridization solutioncontaining 10% dextrane sulfate, 1 M NaCl, 1% SDS and 5×10⁶ cpm ³²plabeled probe, at 65° C., with a final wash solution of 0.2× SSC and0.1% SDS and final wash at 65° C.; (iv) a man induced variation of theportion; or (v) a naturally occurring variation of the portion.

[0024] According to still further features in the described preferredembodiments the polypeptide is at least 70% homologous to SEQ ID NO:3 ora portion thereof having the bicarbonate transporter activity asdetermined using the Blast software where gap open penalty equals 11,gap extension penalty equals 1 and matrix is blosum62.

[0025] According to still further features in the described preferredembodiments the photosynthetic organism is a plant.

[0026] According to still further features in the described preferredembodiments the plant is a C3 plant.

[0027] According to still further features in the described preferredembodiments the C3 plant is selected from the group consisting oftobacco, tomato, soybeans, potato, cucumber, cotton, wheat, rice andbarley.

[0028] According to still further features in the described preferredembodiments the plant is a C4 plant.

[0029] According to still further features in the described preferredembodiments the C4 plant is selected from the group consisting of corn,sugar cane and sorghum.

[0030] According to still further features in the described preferredembodiments the organism is characterized by a photosynthetic rate atleast 10% higher as compared to a control non-transformed organism underotherwise identical conditions.

[0031] According to still further features in the described preferredembodiments the plant promoter is selected from the group consisting ofa constitutive plant promoter, a tissue specific plant promoter and aninducible plant promoter.

[0032] According to still further features in the described preferredembodiments (i) the constitutive plant promoter is independentlyselected from the group consisting of CaMV35S plant promoter, CaMV19Splant promoter, FMV34S plant promoter, sugarcane bacilliform badnavirusplant promoter, CsVMW plant promoter, Arabidopsis ACT2/ACT8 actin plantpromoter, Arabidopsis ubiquitin UBQ1 plant promoter, barley leaf thioninBTH6 plant promoter, and rice actin plant promoter; (ii) the tissuespecific plant promoter is independently selected from the groupconsisting of bean phaseolin storage protein plant promoter, DLEC plantpromoter, PHSβ plant promoter, zein storage protein plant promoter,conglutin gamma plant promoter from soybean, AT2S 1 gene plant promoter,ACT 11 actin plant promoter from Arabidopsis, napA plant promoter fromBrassica napus and potato patatin gene plant promoter; and (iii) theinducible plant promoter is independently selected from the groupconsisting of a light-inducible plant promoter derived from the pea rbcSgene, a plant promoter from the alfalfa rbcS gene, DRE, MYC and MYBplant promoters which are active in drought; INT, INPS, prxEa, Hahsp17.7G4 and RD21 plant promoters active in high salinity and osmoticstress, and hsr203J and str246C plant promoters active in pathogenicstress.

[0033] According to still further features in the described preferredembodiments the polynucleotide further includes a sequence elementselected from the group consisting of a nucleic acid sequence encoding atransit peptide, an origin of replication for propagation in bacterialcells, at least one sequence element for integration into a plant'sgenome, a polyadenylation recognition sequence, a transcriptiontermination signal, a sequence encoding a translation start site, asequence encoding a translation stop site, plant RNA virus derivedsequences, plant DNA virus derived sequences, tumor inducing (Ti)plasmid derived sequences and a transposable element derived sequence.

[0034] The present invention provides new horizons with respect to cropyields, especially of C3 plants grown under low CO₂ and/or under limitedwater availability, because it accelerates the transport of bicarbonateinto plant cells and eventually the chloroplasts to thereby improve thephotosynthetic process and as a result carbon fixation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The invention is herein described, by way of example only, withreference to the accompanying drawings, wherein:

[0036]FIG. 1 is a schematic representation of a genomic region inSynechococcus sp. PCC 7942 where an insertion (indicated by a star) ofan inactivation library fragment led to the formation of mutant IL-2.DNA sequence is available in the GenBank, Accession number U62616.Restriction sites are marked as: A—ApaI, B—BamHI, Ei—EcoRI, E—EcoRV,H—HincII, Hi—HindIII, K—KpnI, M—MfeI, N—NheI, T—TaqI. Underlined lettersrepresent the terminate position of the DNA fragments that were used asprobes. Relevant fragments isolated from an EMBL3 library are marked E1,E2 and E3. P1 and P2 are fragments obtained by PCR. Triangles indicatesites where a cartridge encoding Kan^(r) was inserted. Open readingframes are marked by an arrow and their similarities to other proteinsare noted. Sll and slr (followed by four digits) are the homologousgenes in Synechocystis PCC 6803 [23]; YZ02-myctu, Accession No. Q10536;ICC, Accession No. P36650; Y128-SYNP6, Accession No. P05677; YGGH,Accession No. P44648; Ribosome binding factor A homologous to sll0754and to P45141; O-acetylhomoserine sulfhydrylase homologous to sll0077and NifS. ORF280 started upstream of the schematic representationpresented herein.

[0037]FIG. 2 shows nucleic acid sequence alignment between ORF467 (ICTB,SEQ ID NO:2) and slr1515 (SLR, SEQ ID NO:4). Vertical lines indicatenucleotide identity. Gaps are indicated by hyphens. Alignment wasperformed using the Blast software where gap penalty equals 10 forexistence and 10 for extension, average match equals 10 and averagemismatch equals -5. Identical nucleotides equals 56%.

[0038]FIG. 3 shows amino acid sequence alignment between the IctBprotein (ICTB, SEQ ID NO:3) and the protein encoded by slr1515 (SLR, SEQID NO:5). Identical amino acids are marked by their single letter codebetween the aligned sequences, similar amino acids are indicated by aplus sign. Alignment was performed using the Blast software where gapopen penalty equals 11, gap extension penalty equals 1 and matrix isblosum62. Identical amino acids equals 47%, similar amino acids equals16%, total homology equals 63%.

[0039]FIGS. 4a-b are graphs showing the rates of CO₂ and of HCO₃ ⁻uptake by Synechococcus PCC 7942 (2a) and mutant IL-2 (2b) as a functionof external Ci concentration. The rates were assessed from measurementsduring steady state photosynthesis using a membrane inlet massspectrometer (MIMS) [6, 7, 22].

[0040]FIG. 5 presents DNA sequence homology comparison of a region ofictB found in Synechococcus PCC 7942 and in mutant IL-2. This region wasduplicated in the mutant due to a single cross-over event. Compared withthe wild type, one additional nucleotide and a deletion of sixnucleotides were found in the BamHI side, and 4 nucleotides were deletedin the ApaI side (see FIG. 1). These changes resulted in stop codons inIctB after 168 or 80 amino acids in the BamHI and ApaI sides,respectively. The sequence shown by this Figure starts from the 69^(th)amino acid of ictB.

[0041]FIG. 6 is a photograph of a RNA gel blot showing expression ofictB RNA in transgenic Arabidopsis plants. RNA was isolated fromwild-type (WT) and 3 independent transgenic plants and subjected to RNAgel blot analysis with a probe for ictB. WT plants were found not tocontain transcript(s) that hybridized with the ictB probe. Thetransgenic lines shown in this Figure were used for the CO₂ exchangeanalysis presented in Table 2.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present invention is of a method which can be used to enhanceinorganic carbon fixation by photosynthetic organisms. The presentinvention is further of nucleic acid molecules which can be used toenhance inorganic carbon fixation by photosynthetic organisms. Thepresent invention is further of transformed plants characterized byenhanced inorganic carbon fixation. More specifically, the presentinvention employs an overexpressed bicarbonate transporter for enhancingcarbon fixation in particular C3 plants.

[0043] The principles and operation of the present invention may bebetter understood with reference to the drawings and accompanyingdescriptions.

[0044] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement of thecomponents set forth in the following description or illustrated in thedrawings. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

[0045] According to one aspect of the present invention there isprovided a method of enhancing inorganic carbon fixation by aphotosynthetic organism.

[0046] As used herein in the specification and in the claims sectionthat follows the phrase “photosynthetic organism” includes organisms,both unicellular or multicellular, both prokaryotes or eukaryotes, bothsoil grown or aquatic, capable of producing complex organic materials,especially carbohydrates, from carbon dioxide using light as the sourceof energy and with the aid of chlorophyll and optionally associatedpigments.

[0047] The method according to the present invention is effected bytransforming cells of the photosynthetic organism with an expressiblepolynucleotide encoding a polypeptide having a bicarbonate (HCO₃ ⁻)transporter activity.

[0048] As used herein in the specification and in the claims sectionthat follows the term “transform” and its conjugations such astransformation, transforming and transformed, all relate to the processof introducing heterologous nucleic acid sequences into a cell or anorganism. The term thus reads on, for example, “genetically modified”,“transgenic” and “transfected” or “viral infected” and theirconjugations, which may be used herein to further described the presentinvention. The term relates both to introduction of a heterologousnucleic acid sequence into the genome of an organism and/or into thegenome of a nucleic acid containing organelle thereof, such as into agenome of chloroplast or a mitochondrion.

[0049] As used herein in the specification and in the claims sectionthat follows the phrase “expressible polynucleotide” refers to a nucleicacid sequence including a promoter sequence and a downstream polypeptideencoding sequence, the promoter sequence is so positioned andconstructed so as to direct transcription of the downstream polypeptideencoding sequence.

[0050] As used herein in the specification and in the claims sectionthat follows the term “polypeptide” refers also to a protein, inparticular a transmembrane protein, which may include a transit peptide,and further to a post translationally modified protein, such as, but notlimited to, a phosphorylated protein, glycosylated protein,ubiquitinylated protein, acetylated protein, methylated protein, etc.

[0051] As used herein in the specification and in the claims sectionthat follows the phrase “bicarbonate transporter activity” refers to thedirect activity of a membrane integrated protein in transportingbicarbonate across a membrane in which it is integrated. Such a membranecan be the cell membrane and/or a membrane of an organelle, such as thechloroplast's outer and inner membrane. Such activity can be effected bydirect expenditure of energy, i.e., ATP hydrolysis, which is availableboth in the cytoplasm and the chloroplast's stroma, or by co- oranti-transport, as effected by co- or antiporters while dissipating aconcentration gradient of an ion across a membrane.

[0052] According to another aspect of the present invention there isprovided a nucleic acid molecule for enhancing inorganic carbon fixationby a photosynthetic organism. The nucleic acid molecule according tothis aspect of the present invention includes a polynucleotide encodinga polypeptide having a bicarbonate transporter activity.

[0053] As used herein in the specification and in the claims sectionthat follows the term “nucleic acid molecule” includes polynucleotides,constructs and vectors. The terms “construct” and “vector” may be usedherein interchangeably.

[0054] Such nucleic acid molecule or polynucleotide can be a nucleicacid sequence corresponding to at least a portion derived from SEQ IDNO:2, the portion encodes the protein having the bicarbonate transporteractivity.

[0055] Alternatively or in addition it can be a nucleic acid sequence atleast 60%, preferably at least 65%, more preferably at least 70%, stillmore preferably at least 75%, yet more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, yet morepreferably at least 95%, ideally 95-100% identical to that portion, asdetermined using the Blast software where gap penalty equals 10 forexistence and 10 for extension, average match equals 10 and averagemismatch equals -5. It will be appreciated in this respect that SEQ IDNO:2 can be readily used to isolate homologous sequences which can betested as described in the Examples section that follows for theirbicarbonate transport activity. Methods for isolating such homologoussequences are extensively described in, for example, Sambrook et al. [9]and may include hybridization and PCR amplification.

[0056] Still alternatively or in addition it can be a nucleic acidsegment hybridizable with that portion. Hybridization for long nucleicacids (e.g., above 200 bp in length) is effected according to preferredembodiments of the present invention by stringent or moderatehybridization, wherein stringent hybridization is effected by ahybridization solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDSand 5×10⁶ cpm 32p labeled probe, at 65° C., with a final wash solutionof 0.2× SSC and 0.1% SDS and final wash at 65° C.; whereas moderatehybridization is effected by a hybridization solution containing 10%dextrane sulfate, 1 M NaCl, 1% SDS and 5×10⁶ cpm ³²p labeled probe, at65° C., with a final wash solution of 1× SSC and 0.1% SDS and final washat 50° C.

[0057] Yet alternatively or in addition it can be a man inducedvariation, such as site directed or non-specific mutagenesis of thatportion. Methods of man induced variation of nucleic acids are wellknown in the art and are further described in detail in reference [9].

[0058] Still alternatively or in addition it can be a naturallyoccurring variation of that portion. Such variations can becharacterized and such sequence alterations isolated by one ordinarilyskilled in the art using the assays and procedures described hereinunderin the Examples section that follows.

[0059] According to a preferred embodiment of the present invention thepolypeptide encoded by the polynucleotide is at least 60%, preferably atleast 65%, more preferably at least 70%, still more preferably at least75 %, yet more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, yet more preferably at least 95%, ideally95-100% homologous (identical +similar) to SEQ ID NO:3 or a portionthereof having the bicarbonate transporter activity as determined usingthe Blast software where gap open penalty equals 11, gap extensionpenalty equals 1 and matrix is blosum62.

[0060] According to a preferred embodiment of the present invention, thepolypeptide includes an N terminal transit peptide fused thereto whichserves for directing the polypeptide to a specific membrane. Such amembrane can be, for example, the cell membrane, wherein the polypeptidewill serve to transport bicarbonate from the apoplast into thecytoplasm, or, such a membrane can be the outer and preferably the innerchloroplast membrane, wherein the polypeptide will serve to transportbicarbonate from the cytoplasm to the intermembranal space and thestroma, respectively. Transit peptides which function as hereindescribed are well known in the art. Further description of such transitpeptides is found in, for example, Johnson et aL The Plant Cell (1990)2:525-532; Sauer et al. EMBO J. (1990) 9:3045-3050; Mueckler et al.Science (1985) 229:941-945; Von Heijne, Eur. J. Biochem. (1983)133:17-21; Yon Heijne, J. Mol. Biol. (1986) 189:239-242; Iturriaga etal. The Plant Cell (1989) 1:381-390; McKnight et al., Nucl. Acid Res.(1990) 18:4939-4943; Matsuoka and Nakamura, Proc. Natl. Acad. Sci. USA(1991) 88:834-838. A recent text book entitled “Recombinant proteinsfrom plants”, Eds. C. Cunningham and A. J. R. Porter, 1998 Humana PressTotowa, N.J. describe methods for the production of recombinant proteinsin plants and methods for targeting the proteins to differentcompartments in the plant cell. The book by Cunningham and Porter isincorporated herein by reference. It will however be appreciated by oneof skills in the art that a large number of membrane integrated proteinsfail to poses a removable transit peptide. It is accepted that in suchcases a certain amino acid sequence in said proteins serves not only asa structural portion of the protein, but also as a transit peptide.

[0061] According to a preferred embodiment, the nucleic acid moleculefurther includes a plant promoter located upstream to the polynucleotideand being effective in expressing the polypeptide.

[0062] As used herein in the specification and in the claims sectionthat follows the phrase “plant promoter” includes a promoter which candirect gene expression in plant cells (including DNA containingorganelles). Such a promoter can be derived from a plant, bacterial,viral, fungal or animal origin. Such a promoter can be constitutive,i.e., capable of directing high level of gene expression in a pluralityof plant tissues, tissue specific, i.e., capable of directing geneexpression in a particular plant tissue or tissues, inducible, i.e.,capable of directing gene expression under a stimulus, or chimeric.

[0063] Thus, the plant promoter employed can be a constitutive promoter,a tissue specific promoter, an inducible promoter or a chimericpromoter.

[0064] Examples of constitutive plant promoters include, without beinglimited to, CaMV35S and CaMV19S promoters, FMV34S promoter, sugarcanebacilliform badnavirus promoter, CsVMV promoter, Arabidopsis ACT2/ACT8actin promoter, Arabidopsis ubiquitin UBQ1 promoter, barley leaf thioninBTH6 promoter, and rice actin promoter.

[0065] Examples of tissue specific promoters include, without beinglimited to, bean phaseolin storage protein promoter, DLEC promoter, PHSβpromoter, zein storage protein promoter, conglutin gamma promoter fromsoybean, AT2S1 gene promoter, ACT 11 actin promoter from Arabidopsis,napA promoter from Brassica napus and potato patatin gene promoter.

[0066] The inducible promoter is a promoter induced by a specificstimuli such as stress conditions comprising, for example, light,temperature, chemicals, drought, high salinity, osmotic shock, oxidantconditions or in case of pathogenicity and include, without beinglimited to, the light-inducible promoter derived from the pea rbcS gene,the promoter from the alfalfa rbcS gene, the promoters DRE, MYC and MYBactive in drought; the promoters INT, INPS, prxea, Ha hsp17.7G4 and RD21active in high salinity and osmotic stress, and the promoters hsr203Jand str246C active in pathogenic stress.

[0067] There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledenous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276). The principle methods of causing stableintegration of exogenous DNA into plant genomic DNA include two mainapproaches:

[0068] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987)Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Cultureand Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of PlantNuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers,San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

[0069] (ii) direct DNA uptake: Paszkowski et al., in Cell Culture andSomatic Cell Genetics of Plants, Vol. 6, Molecular Biology of PlantNuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers,San Diego, Calif. (1989) p. 52-68; including methods for direct uptakeof DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology6:1072-1074. DNA uptake induced by brief electric shock of plant cells:Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature(1986) 319:791-793. DNA injection into plant cells or tissues byparticle bombardment, Klein et al. Bio/Technology (1988) 6:559-563;McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant.(1990) 79:206-209; by the use of micropipette systems: Neuhaus et al,Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol.Plant. (1990) 79:213-217; or by the direct incubation of DNA withgerminating pollen, DeWet et aL in Experimental Manipulation of OvuleTissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman,London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986)83:715-719.

[0070] The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et aL in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementaryapproach employs the Agrobacterium delivery system in combination withvacuum infiltration. The Agrobacterium system is especially viable inthe creation of transgenic dicotyledenous plants.

[0071] There are various methods of direct DNA transfer into plantcells. In electroporation, the protoplasts are briefly exposed to astrong electric field. In microinjection, the DNA is mechanicallyinjected directly into the cells using very small micropipettes. Inmicroparticle bombardment, the DNA is adsorbed on microprojectiles suchas magnesium sulfate crystals or tungsten particles, and themicroprojectiles are physically accelerated into cells or plant tissues.

[0072] Following transformation plant propagation is exercised. The mostcommon method of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. Therefore, itis preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

[0073] Micropropagation is a process of growing new generation plantsfrom a single piece of tissue that has been excised from a selectedparent plant or cultivar. This process permits the mass reproduction ofplants having the preferred tissue expressing the fusion protein. Thenew generation plants which are produced are genetically identical to,and have all of the characteristics of, the original plant.Micropropagation allows mass production of quality plant material in ashort period of time and offers a rapid multiplication of selectedcultivars in the preservation of the characteristics of the originaltransgenic or transformed plant. The advantages of cloning plants arethe speed of plant multiplication and the quality and uniformity ofplants produced.

[0074] Micropropagation is a multi-stage procedure that requiresalteration of culture medium or growth conditions between stages. Thus,the micropropagation process involves four basic stages: Stage one,initial tissue culturing; stage two, tissue culture multiplication;stage three, differentiation and plant formation; and stage four,greenhouse culturing and hardening. During stage one, initial tissueculturing, the tissue culture is established and certifiedcontaminant-free. During stage two, the initial tissue culture ismultiplied until a sufficient number of tissue samples are produced tomeet production goals. During stage three, the tissue samples grown instage two are divided and grown into individual plantlets. At stagefour, the transformed plantlets are transferred to a greenhouse forhardening where the plants' tolerance to light is gradually increased sothat it can be grown in the natural environment.

[0075] The basic bacterial/plant vector construct will preferablyprovide a broad host range prokaryote replication origin; a prokaryoteselectable marker; and, for Agrobacterium transformations, T DNAsequences for Agrobacterium-mediated transfer to plant chromosomes.Where the heterologous sequence is not readily amenable to detection,the construct will preferably also have a selectable marker genesuitable for determining if a plant cell has been transformed. A generalreview of suitable markers for the members of the grass family is foundin Wilmink and Dons, Plant Mol. Biol. Reptr. (1993)11:165-185.

[0076] Sequences suitable for permitting integration of the heterologoussequence into the plant genome are also recommended. These might includetransposon sequences and the like for homologous recombination as wellas Ti sequences which permit random insertion of a heterologousexpression cassette into a plant genome.

[0077] Suitable prokaryote selectable markers include resistance towardantibiotics such as ampicillin or tetracycline. Other DNA sequencesencoding additional functions may also be present in the vector, as isknown in the art.

[0078] The constructs of the subject invention will include anexpression cassette for expression of the fusion protein of interest.Usually, there will be only one expression cassette, although two ormore are feasible. The recombinant expression cassette will contain inaddition to the heterologous sequence one or more of the followingsequence elements, a promoter region, plant 5′ untranslated sequences,initiation codon depending upon whether or not the structural gene comesequipped with one, and a transcription and translation terminationsequence. Unique restriction enzyme sites at the 5′ and 3′ ends of thecassette allow for easy insertion into a pre-existing vector.

[0079] Viruses are a unique class of infectious agents whose distinctivefeatures are their simple organization and their mechanism ofreplication. In fact, a complete viral particle, or virion, may beregarded mainly as a block of genetic material (either DNA or RNA)capable of autonomous replication, surrounded by a protein coat andsometimes by an additional membranous envelope such as in the case ofalpha viruses. The coat protects the virus from the environment andserves as a vehicle for transmission from one host cell to another.

[0080] Viruses that have been shown to be useful for the transformationof plant hosts include CaV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

[0081] Construction of plant RNA viruses for the introduction andexpression of non-viral foreign genes in plants is demonstrated by theabove references as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; and Takamatsu et aL FEBS Letters (1990)269:73-76.

[0082] When the virus is a DNA virus, the constructions can be made tothe virus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

[0083] Construction of plant RNA viruses for the introduction andexpression of non-viral foreign genes in plants is demonstrated by theabove references as well as in U.S. Pat. No. 5,316,931.

[0084] In one embodiment, a plant viral nucleic acid is provided inwhich the native coat protein coding sequence has been deleted from aviral nucleic acid, a non-native plant viral coat protein codingsequence and a non-native promoter, preferably the subgenomic promoterof the non-native coat protein coding sequence, capable of expression inthe plant host, packaging of the recombinant plant viral nucleic acid,and ensuring a systemic infection of the host by the recombinant plantviral nucleic acid, has been inserted. Alternatively, the coat proteingene may be inactivated by insertion of the non-native nucleic acidsequence within it, such that a fusion protein is produced. Therecombinant plant viral nucleic acid may contain one or more additionalnon-native subgenomic promoters. Each non-native subgenomic promoter iscapable of transcribing or expressing adjacent genes or nucleic acidsequences in the plant host and incapable of recombination with eachother and with native subgenomic promoters. Non-native (foreign) nucleicacid sequences may be inserted adjacent the native plant viralsubgenomic promoter or the native and a non-native plant viralsubgenomic promoters if more than one nucleic acid sequence is included.The non-native nucleic acid sequences are transcribed or expressed inthe host plant under control of the subgenomic promoter to produce thedesired products.

[0085] In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

[0086] In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that said sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

[0087] In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

[0088] The viral vectors are encapsidated by the coat proteins encodedby the recombinant plant viral nucleic acid to produce a recombinantplant virus. The recombinant plant viral nucleic acid or recombinantplant virus is used to infect appropriate host plants. The recombinantplant viral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)in the host to produce the desired fusion protein.

[0089] Thus, according to a preferred embodiment of the presentinvention the polynucleotide or nucleic acid molecule of the presentinvention further includes one or more sequence elements, such as, butnot limited to, a nucleic acid sequence encoding a transit peptide, anorigin of replication for propagation in bacterial cells, at least onesequence element for integration into a plant's genome, apolyadenylation recognition sequence, a transcription terminationsignal, a sequence encoding a translation start site, a sequenceencoding a translation stop site, plant RNA virus derived sequences,plant DNA virus derived sequences, tumor inducing (Ti) plasmid derivedsequences and a transposable element derived sequence.

[0090] According to another preferred embodiment of the presentinvention, the step of transforming the cells of the photosyntheticorganism with the expressible polynucleotide encoding the bicarbonatetransporter is effected by a method such as genetic transformation andtransient transformation. Genetic transformation can be effected by, forexample, Agrobaterium mediated transformation, whereas transienttransformation can be effected by, for example, viral transformation.Both transient and genetic transformation can be effected byelectroporation, particle bombardment or any of the other methods listedand further described hereinabove.

[0091] A technique for introducing heterologous nucleic acid sequencesto the genome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theheterologous nucleic acid is introduced via particle bombardment intothe cells with the aim of introducing at least one heterologous nucleicacid molecule into the chloroplasts. The heterologous nucleic acid isselected such that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the heterologous nucleic acid includes,in addition to a gene of interest, at least one nucleic acid stretchwhich is derived from the chloroplast's genome. In addition, theheterologous nucleic acid includes a selectable marker, which serves bysequential selection procedures to ascertain that all or substantiallyall of the copies of the chloroplast genomes following such selectionwill include the heterologous nucleic acid. Further details relating tothis technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507which are incorporated herein by reference. A polypeptide can thus beproduced by the protein expression system of the chloroplast and becomeintegrated into the chloroplast's inner membrane. It will be appreciatedthat cyanobacterial proteins are expected to express well by thechloroplast expression system.

[0092] According to another aspect of the present invention there isprovided a transformed photosynthetic organism comprising the nucleicacid molecule or polynucleotide described herein.

[0093] According to a preferred embodiment of the present invention thephotosynthetic organism is a plant, such as a crop plant. Preferably theplant is a C3 plant, such as, but not limited to, tobacco, tomato,soybeans, potato, cucumber, cotton, wheat, rice and barley because, asfurther detailed herein, such plants are more sensistive to limitedwater supply and/or low CO₂ atmosphere. However, over expressing C4plants, such as, but not limited to, corn, sugar cane and sorghum arealso expected to enjoy improved CO₂ fixation, especially underwater-limiting conditions.

[0094] According to a preferred embodiment of the present invention thetransformed photosynthetic organism is characterized by a photosyntheticrate at least 5%, preferably at least 10%, more preferably at least 15%,yet more preferably at least 25%, still more preferably at least 30%,more preferably at least 40%, more preferably at least 50%, ideally morepreferably at least between 50% and 100% higher as compared to a controlnon-transformed organism under otherwise identical conditions. suchconditions and photosynthesis rate measurement procedures are furtherdescribed in the Examples section that follows with respect to Table 2.

[0095] Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

[0096] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

[0097] Generally, the nomenclature used herein and the laboratoryprocedures in recombinant DNA technology described below are those wellknown and commonly employed in the art. Standard techniques are used forcloning, DNA and RNA isolation, amplification and purification.Generally enzymatic reactions involving DNA ligase, DNA polymerase,restriction endonucleases and the like are performed according to themanufacturers' specifications. These techniques and various othertechniques are generally performed according to Sambrook et al. [9].Other general references are provided throughout this document. Theprocedures therein are believed to be well known in the art and areprovided for the convenience of the reader. All the informationcontained therein is incorporated herein by reference.

Materials and Experimental Methods

[0098] Growth conditions:

[0099] Cultures of Synechococcus sp. strain PCC 7942 and mutant IL-2thereof were grown at 30° C. in BG₁₁ medium supplemented with 20 mMHepes-NaOH pH 7.8 and 25 μg mL⁻¹ kanamycin (in the case of the mutant).The medium was aerated with either 5% v/v CO₂ in air (high CO₂) or0.0175% v/v CO₂ in air (low CO₂) which was prepared by mixing air withCO₂-free air at a 1:1 ratio. Escherichia coil (strain DH5α) were grownon an LB medium [9] supplemented with either kanamycin (50 μg/mL) orampicillin (50 μg/mL) when required.

[0100] Measurements of Photosynthesis and Ci Uptake:

[0101] The rates of inorganic carbon (Ci)-dependent O₂ evolution weremeasured by an O₂ electrode as described elsewhere [10] and by amembrane inlet mass spectrometer (MIMS, [6, 11]). The MIMS was also usedfor assessments of CO₂ and HCO₃ ⁻ uptake during steady statephotosynthesis [6]. Ci fluxes following supply of CO₂ or HCO₃ ⁻ weredetermined by the filtering centrifugation technique [10]. High-CO₂grown cells in the log phase of growth were transferred to either low orhigh CO₂ 12 hours before conducting the experiments. Following harvest,the cells were resuspended in 25 mM Hepes-NaOH pH 8.0 and aerated withair (Ci concentration was about 0.4 mM) under light flux of 100 μmolphoton quanta m⁻²s⁻ Aliquots were withdrawn, immediately placed inmicrofuge tubes and kept under similar light and temperature conditions.Small amounts of ¹⁴C—CO₂ or ¹⁴C—HCO₃ ⁻ which did not affect the final Ciconcentration, were injected, and the Ci uptake terminated after 5seconds by centrifugation.

[0102] General DNA Manipulations:

[0103] Genomic DNA was isolated as described elsewhere [12]. Standardrecombinant DNA techniques were used for cloning and Southern analyses[12-13] using the Random Primed DNA Labeling Kit or the DIG system,(Boehringer, Mannheim). Sequence analysis was performed using the DyeTerminator cycle sequencing kit, ABI Prism (377 DNA sequencing PerkinElmer). The genomic library used here was constructed using LambdaEMBL3/BamHI vector kit (Stratagene, La Jolla, Calif.).

[0104] Construction and Isolation of Mutant IL-2:

[0105] A modification of the method developed by Dolganov and Grossman[14] was used to raise and isolate new high-CO₂-requiring mutants [4,5]. Briefly, genomic DNA was digested with TaqI and ligated into theAccI site of the polylinker of a modified Bluescript SK plasmid. Thebluescript borne gene for conferring ampicillin resistance (within theScal site) was inactivated by the insertion of a cartridge encodingkanamycin resistance (Kan^(r), [8]). Synechococcus sp. train PCC 7942cells were transfected with the library [12]. Single crossover eventswhich conferred Kan^(r) led to inactivation of various genes. TheKan^(r) cells were exposed to low CO₂ conditions for 8 hours foradaptation, followed by an ampicillin treatment (400 g/mL) for 12 hours.Cells capable of adapting to low CO₂ and thus able to grow under theseconditions were eliminated by this treatment. The high-CO₂-requiringmutant, IL-2, unable to divide under low CO₂ conditions, survived, andwas rescued following the removal of ampicillin and growth in thepresence of high CO₂ concentration.

[0106] Cloning of the Relevant Impaired Genomic Region from Mutant IL-2:

[0107] DNA isolated from the mutant was digested with ApaI located onone side of the AccI site in the polylinker; with BamHI or EcoRI,located on the other side of the AccI site; or with MfeI that does notcleave the vector or the Kan^(r) cartridge. These enzymes also cleavedthe genomic DNA. The digested DNA was self-ligated followed bytransfection of competent E. coli cells (strain DH5α). Kan^(r) coloniescarrying the vector sequences bearing the origin of replication, theKan^(r) cartridge and part of the inactivated gene were then isolated.This procedure was used to clone the flanking regions on both sides ofthe vector inserted into the mutant. A 1.3 Kbp ApaI and a 0.8 Kbp BamHIfragments isolated from the plasmids (one ApaI site and BamHI siteoriginated from the vector's polylinker) were used as probes to identifythe relevant clones in an EMBL3 genomic library of a wild type genome,and for Southern analyses. The location of these fragments in the wildtype genome (SEQ ID NO:1) is schematically shown in FIG. 1. The ApaIfragment is between positions 1600 to 2899 (SEQ ID NO:1), marked as Tand A in FIG. 1; the BamHI fragment is between positions 4125 to 4957(SEQ ID NO: 1) marked as B and T in FIG. 1. The 0.8 Kbp BamHI fragmenthybridized with the 1.6 Kbp HincII fragment (marked E3 in FIG. 1). The1.3 Kbp ApaI fragment hybridized with an EcoRI fragment of about 6 Kbp.Interestingly, this fragment could not be cloned from the genomiclibrary into E. coli. Therefore, the BamHI site was used (position 2348,SEQ ID NO:1, FIG. 1) to split the EMBL3 clone into two clonablefragments of 4.0 and 1.8 Kbp (E1 and E2, respectively, E1 starts from aSau3A site upstream of the HindIII site positioned at the beginning ofFIG. 1). Confirmation that these three fragments were indeed located asshown in FIG. 1 was obtained by PCR using wild type DNA as template,leading to the synthesis of fragments PI and P2 (FIG. 1). Sequenceanalyses enabled comparison of the relevant region in IL-2 with thecorresponding sequence in the wild-type.

Experimental Results

[0108] Physiological analysis:

[0109] The IL-2 mutant grew nearly the same as the wild type cells inthe presence of high CO₂ concentration but was unable to grow under lowCO₂. Analysis of the photosynthetic rate as a function of external Ciconcentration revealed that the apparent photosynthetic affinity of theIL-2 mutant was 20 mM Ci, which is about 100 times higher than theconcentration of Ci at the low CO₂ conditions. The curves relating tothe photosynthetic rate as a function of Ci concentration, in IL-2, weresimilar to those obtained with other high-CO₂-requiring mutants ofSynechococcus PCC 7942 [16, 17]. These data suggested that the inabilityof IL-2 to grow under low CO₂ is due to the poor photosyntheticperformance of this mutant.

[0110] High-CO₂-requiring mutants showing such characteristics wererecognized among mutants bearing aberrant carboxysomes [9, 10, 12, 18,19] or defective in energization of Ci uptake [20, 21]. All thecarboxysome-defective mutants characterized to date were able toaccumulate Ci within the cells similarly to wild type cells. However,they were unable to utilize it efficiently in photosynthesis due to lowactivation state of rubisco in mutant cells exposed to low CO₂ [10].This was not the case for mutant IL-2 which possessed normalcarboxysomes but exhibited impaired HCO₃ ⁻ uptake (Table 1, FIGS. 4a-b).Measurements of ¹⁴Ci accumulation indicated that HCO₃ ⁻ and CO₂ uptakewere similar in the high-CO₂-grown wild type and the mutant TABLE 1 CO₂Uptake HCO₃ ⁻ Uptake High CO₂ Low CO₂ High CO₂ Low CO₂ WT 31.6 53.9 30.9182.0 IL-2 26.6 39.2 32.2 61.1

[0111] Uptake of HCO₃ ⁻ by wild type cells increased by approximately6-fold following exposure to low CO₂ conditions for 12 hours. On theother hand, the same treatment resulted in only up to a 2-fold increasein HCO₃ ⁻ uptake for the IL-2 mutant. Uptake of CO₂ increased byapproximately 50% for both the wild type and the IL-2 mutant followingtransfer from high- to low CO₂ conditions. These data indicate that HCO₃⁻ transport and not CO₂ uptake was impaired in mutant IL-2.

[0112] The V_(max) of HCO₃ ⁻ uptake, estimated by MIMS [7, 22] at steadystate photosynthesis (FIG. 4a), were 220 and 290 μmol HCO₃ ⁻ mg⁻¹ Chlh⁻¹ for high- and low-CO₂-grown wild type, respectively, and thecorresponding K_(½) (HCO₃ ⁻ ) were 0.3 and 0.04 mM HCO₃ ⁻, respectively.These estimates are in close agreement with those reported earlier [7].In high-CO₂-grown mutant IL-2, on the other hand, the HCO₃ ⁻transporting system was apparently inactive. The curve relating the rateof HCO₃ transport as a function of its concentration did not resemblethe expected saturable kinetics (observed for the wild type), but wascloser to a linear dependence as expected in a diffusion mediatedprocess (FIG. 4b). It was essential to raise the concentration of HCO₃ ⁻in the medium to values as high as 25 mM in order to achieve rates ofHCO₃ ⁻ uptake similar to the V_(max) depicted by the wild type.

[0113] The estimated V_(max) of CO₂ uptake by high-CO₂-grown wild typeand IL-2 was similar for both at around 130-150 μmol CO₂ mg⁻¹ Chl h⁻¹and the K_({fraction (2)}) (CO₂) values were around 5 μM (FIGS. 4a-b),indicating that CO₂ uptake was far less affected by the mutation inIL-2. Mutant cells that were exposed to low CO₂ for 12 hours showedsaturable kinetics for HCO₃ ⁻ uptake suggesting the involvement of acarrier. However, the K_(½) (HCO₃ ⁻) was 4.5 mM HCO₃ (i.e., 15- and100-fold lower than in high- and in low-CO₂-grown wild type,respectively) and the V_(max) was approximately 200 μmol HCO₃ ⁻ mg⁻¹ Chlh⁻¹. These data indicate the presence of a low affinity HCO₃ ⁻transporter that is activated or utilized following inactivation of ahigh affinity HCO₃ ⁻ uptake in the mutant. The activity of the lowaffinity transporter resulted in the saturable transport kineticsobserved in the low-CO₂-exposed mutant. These data further demonstratedthat the mutant was able to respond to the low CO₂ signal.

[0114] The reason for the discrepancy between the data obtained by thetwo methods used, with respect to HCO₃ ⁻ uptake in wild type and mutantcells grown under high-CO₂-conditions, is not fully understood. It mightbe related to the fact that in the MIMS method HCO₃ ⁻ uptake is assessedas the difference between net photosynthesis and CO₂ uptake [6, 7, 22].Therefore, at Ci concentrations below 3 mM, where the mutant did notexhibit net photosynthesis, HCO₃ ⁻ uptake was calculated as zero (FIGS.4a-b). On the other hand, the filtering centrifugation technique, asused herein, measured the unidirectional HCO₃ ⁻ transport close tosteady state via isotope exchange, which can explain some of thevariations in the results. Not withstanding, the data obtained by bothmethods clearly indicates severe inhibition of HCO₃ ⁻ uptake in mutantcells exposed to low CO₂. It is interesting to note that while thecharacteristics of HCO₃ ⁻ uptake changed during acclimation of themutant to low CO₂, CO₂ transport was not affected (FIGS. 4a-b). It isthus concluded that the high-CO₂-requiring phenotype of IL-2 isgenerated by the mutation of a HCO₃ transporter rather than innon-acclimation to low CO₂.

[0115] Genomic Analysis:

[0116] Since IL-2 is impaired in HCO₃ ⁻ transport, it was used toidentify and clone the relevant genomic region involved in the highaffinity HCO₃ ⁻ uptake. FIG. 1 presents a schematic map of the genomicregion in Synechococcus sp. PCC 7942 where the insertion of theinactivating vector by a single cross over recombination event(indicated by a star) generated the IL-2 mutant. Sequence analysis(GenBank, accession No. U62616, SEQ ID NO:1) identified several openreading frames (identified in the legend of FIG. 1), some are similar tothose identified in Synechocystis PCC 6803 [23]. Comparison of the DNAsequence in the wild type with those in the two repeated regions (due tothe single cross over) in mutant IL-2, identified several alterations inthe latter. This included a deletion of 4 nucleotides in the ApaI sideand a deletion of 6 nucleotides but the addition of one bp in the BamHIside (FIG. 5). The reason(s) for these alterations is not known, butthey occurred during the single cross recombination between the genomicDNA and the supercoiled plasmid bearing the insert in the inactivationlibrary. The high-CO₂-requiring phenotype of mutant JR12 ofSynechococcus sp. PCC 7942 also resulted from deletions of part of thevector and of a genomic region, during a single cross over event,leading to a deficiency in purine biosynthesis under low CO₂ [24].

[0117] The alterations depicted in FIG. 5 resulted in frame shifts whichled to inactivation of both copies of ORF467 (nucleotides 2670-4073 ofSEQ ID NO:1, SEQ ID NO:2) in IL-2. Insertion of a Kan^(r) cartridgewithin the EcoRV or NheI sites in ORF467, positions 2919 and 3897 (SEQID NO:1), respectively (indicated by the triangles in FIG. 1), resultedin mutants capable of growing in the presence of kanamycin under low CO₂conditions, though significantly (about 50%) slower than the wild type.Southern analyses of these mutants clearly indicated that they weremerodiploids, i.e., contained both the wild type and the mutated genomicregions.

[0118]FIGS. 2 and 3 show nucleic and amino acid alignments of ictB andslr1515, the most similar sequence to ictB identified in the gene bank,respectively. Note that the identical nucleotides shared between thesenucleic acid sequences (FIG. 2) equal 56%, the identical amino acidsshared between these amino acid sequences (FIG. 3) equal 47%, thesimilar amino acids shared between these amino acid sequences (FIG. 3)equal 16%, bringing the total homology therebetween to 63% (FIG. 3).When analyzed without the transmembrane domains, the identical aminoacids shared between these amino acid sequences equal 40%, the similaramino acids shared between these amino acid sequences equal 12%,bringing the total homology therebetween to 52

[0119] HCO₃ ⁻ Transporters in Synechococcus sp. PCC 7942:

[0120] The protein encoded by 0RF467 (SEQ ID NO:3) contains 10 putativetransmembrane regions and is a membrane integrated protein. It issomewhat homologous to several oxidation-reduction proteins includingthe Na⁺/pantothenate symporter of E. coli (Accession No. P16256). Na⁺ions are essential for HCO₃ ⁻ uptake in cyanobacteria and the possibleinvolvement of a Na⁺/HCO₃ ⁻ symport has been discussed [3, 25, 26]. Thesequence of the fourth transmembrane domain contains a region which issimilar to the DCCD binding motif in subunit C of ATP synthase with theexception of the two outermost positions, replaced by conservativechanges in ORF467. The large number of transport proteins that arehomologous to the gene product of ORF467 also suggest that it is also atransport protein, possibly involved in HCO₃ ⁻ uptake. ORF467 isreferred to herein as ictB (for inorganic carbon transport B [27]).

[0121] Sequence similarity between cmpA, encoding a 42-kDa polypeptidewhich accumulates in the cytoplasmic-membrane of low-CO₂-exposedSynechococcus PCC 7942 [28], and nrtA involved in nitrate transport[29], raised the possibility that CmpA may be the periplasmic part of anABC-type transporter engaged in HCO₃ ⁻ transport [21, Omata, personalcommunication]. The role of the 42 kDa polypeptide, however, is notclear since inactivation of cmpA did not affect the ability ofSynechococcus PCC7942 [30] and Synechocystis PCC6803 [21] to grow undera normal air level of CO₂ but growth was decreased under 20 ppm CO₂ inair [21]. It is possible that Synechococcus sp. PCC 7942 contains threedifferent HCO₃ ⁻ carriers: the one encoded by cmpA; IctB; and the oneexpressed in mutant IL-2 cells exposed to low CO₂ whose identity is yetto be elucidated. These transporters enable the cell to maintaininorganic carbon supply under various environmental conditions.

[0122] Transgenic Plants Transformed with ictB:

[0123] The coding region of ictB was cloned downstream of a strongpromoter (CaMV 35S) and down stream to and in frame with the transitpeptide of pea rubisco small subunit within a plasmid bearing a geneencoding kanamycin resistance, and suitable for the Agrobacteriummediated transformation of Arabidopsis. Transformation of Arabidopsiswith this construct resulted in fast growing transgenic plants whichwere kanamycin resistant. Northern analysis performed on wild type andthree transgenic plants (FIG. 6) demonstrated that the transformantsexpressed the ictB whereas, as expected, the wild type did not.

[0124] Measurements of the photosynthetic rate with the aid of a gasexchange system indicated a considerably faster CO₂ fixation in thetransgenic plants (Table 2). The improved rate of CO₂ fixation wasmainly pronounced under low CO₂ concentrations such as experiencedwithin the air spaces in the leaves of crop plants under mostenvironmental conditions. At higher CO₂ concentrations, where the rateof photosynthesis is CO₂-saturated and presumably independent of HCO₃ ⁻transport, the wild type and transgenic plants exhibited similarphotosynthetic rate, as expected. In a typical air space where the CO₂concentration is 200-300 ppm, the photosynthetic rate of the transgenicplant was 24 to 42% higher than in the wild type. At lower CO₂concentrations such as effectively expected under water-limitingconditions the superiority of the transgenic plant was even morepronounced (Table 2). TABLE 2 CO₂ Photosynthetic rate Photosyntheticrate Transgenic Concentration Wild Type Transgenic plant plant/ (ppm)(μmole CO₂ m⁻² s⁻¹) (μmole CO₂ m⁻² s⁻¹) Wild Type 50 0.86 1.35 1.57 1001.72 2.7 1.57 150 2.58 3.69 1.43 200 3.2 4.55 1.42 250 3.93 5.17 1.31300 4.67 5.78 1.24 400 5.78 6.52 1.13 500 6.64 6.89 1.04 600 6.89 7.011.02

[0125] It is thus expected that transgenic C3 plants, such as, but notlimited to, tobacco, tomato, soybeans, potato, cucumber, cotton, wheat,rice, barley and other C3 and also C4 crop plants, such as corn, sugarcane, sohrgum and others, expressing ictB will grow faster, and producehigher crop yield, including, for example, more and heavier fruits,especially under CO₂ and/or water limiting conditions. Tissue specificpromoters, such as leaf specific promoters, could be used to highlyexpress the ictB gene in leaves which are the primary photosyntheticorgans in most plants.

[0126] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

1 9 1 4957 DNA Synechococcus sp. 1 aagcttggat tgaagcgatc ggggtcaatcccagcgatga tcctcagttc ctcctgatgg 60 tcgatccctt tagcgccaag attgaggatctgctgcaagg gctggatttc gcctatcccg 120 aggccgtgaa agtgggcgga ttggccagtggtttgggggc agagtcagcg atcgccagct 180 tgttttttca agaccgacag gtcgatggcgtgattgggct agccctcagt ggcaatgtcc 240 agctgcaggc gatcgtggct cagggctgtcgtccagttgg cccgctttgg catgtggcag 300 cggcggagcg caacattctg cggcaacttcagaccgaaga cgaggaaccg atcgccgcgc 360 tgcaagccct acagtcagtc ctgcgtgatctctcccctga attacagcga tcgctctgtg 420 tgggcctggc ctgcaattct ttccaaacggtattacaacc gggcgacttc ctgatccgta 480 acctgctggg gtttgatccc cgcactggtgctgtagcaat cggcgatcgc attcgagttg 540 ggcagcggct gcagctgcac gtacgggatgcccagacagc ggcggatgac ctcgagcggc 600 aactggggca atggtgccgg cagcatgcgacaaaaccagc agcttccctc ttgttttcct 660 gcttggggcg cggcaagccc ttctatcagcaggccaactt cgagtcgcaa ctgattcagc 720 attacctctc agagctgccc ctagctggctttttctgtaa tggcgaaatc ggcccgatcg 780 ctggcagcac ctacctgcat ggctacacatcggtgctggc tttgctgtcg gccaaaactc 840 actagcgcca gcgagacctg attgtcgatctgctgagcgc gactgtagcg ctggaaatag 900 gcccggacct gagcaggcgc atcggccaagctgaccgtag tatcaccgtc agccaccccc 960 gcccagaaat tccgcaacat cggcaggagagcgatcgcct ccgcctccga taaattcaac 1020 ggctcatggg tcaacaggcg gatcaagtactctgactgcg atcgccatcc attcccgccg 1080 aaaacgtttg taaatcagtc ttgatccggtagcgatcgca cccgacggga ctctagttct 1140 agttgccaac cttcagcggc aggttgtacggttccgagtc ggtagggatg gggatagctg 1200 accaaggaac cggtcgtgac ttcccagagagcaccttgct gactggtggc ttggatgtgg 1260 aggtggcctg tgaagatcac cgagacgctgcccgcttcga ggattgatcg caattcctcg 1320 gcattttcta agatgtagcg ctgaccaagcggatgctgct gttgatcggg cagatgctcc 1380 aacacattgt ggtgaatcat cacccagcgttggctagcgg tggaagtggc gagttcttgt 1440 tgcagccagt tgagttgcgc gcaatcgactcgcccccgat gcagttgatg gcccgcttca 1500 tcaaaagcga tcgaattcag cgcaaacagatcgagatccg gtgcgatcgt gcagcgatag 1560 taggggcgat cgctcgtgaa gccaaagtcttgatagagct cgacaaactc ggccacaccg 1620 gtgcgatcgc gatcgctcgc tgcggcgggcatatcgtggt tgcccggcac cacatagacc 1680 ggatagggca actggcgcaa ttgttgcagcagccactgat ggttttcccg ctccccgtgc 1740 tgggttaaat cccccggcag caacaggaagtccaaatcca gcgctgccag ttctgtcagg 1800 atttgctcaa aagccggaat gctgcactcaatcaaatgga agcgatgggg atggtgccaa 1860 attgtctgcg gcagtccaat gtggagatcgctcagcagcg caaatcgaaa cgctcggttc 1920 attgccatcc cctcagctat cgagcccgattctaggcgaa gctaggtcga gtccgttgtc 1980 ttcagttgca agcattcatg gccagagttcgcgttcggca gcacgtcaat ccgctctctc 2040 agaaattcca agtggtcacg acttggccggattggcaaca ggtctatgcg gactgcgatc 2100 gcccgctgca tttggatatt ggctgtgctcgcgggcgctt tctgctggca atggcgacac 2160 gacaacctga gtggaattat ctggggctggaaattcgtga gccgctggta gatgaggcga 2220 acgcgatcgc ccgcgaacgt gaactgaccaatctctacta ccacttcagc aacgccaatt 2280 tggacttgga accgctgctg cgatcgctgccgacagggat tttgcagcgg gtcagcattc 2340 agttcccgga tccttggttc aagaaacgccatcaaaagcg acgcgtcgtc cagccggaac 2400 tggtgcaagc cctcgcgact gcgttacctgctggtgcaga ggtctttctg caatccgatg 2460 tgctggaagt gcaggcagag atgtgcgaacactttgcggc ggaaccccgc tttcagcgca 2520 cctgcttgga ctggctgccg gaaaatccgctgcccgtccc gaccgagcgc gaaattgccg 2580 ttcaaaacaa acagttgcca gtctaccgtgctctcttcat tcggcagcca gcggactaag 2640 ctcttaaggc aagcgttgac gcgatcgcgatgactgtctg gcaaactctg acttttgccc 2700 attaccaacc ccaacagtgg ggccacagcagtttcttgca tcggctgttt ggcagcctgc 2760 gagcttggcg ggcctccagc cagctgttggtttggtctga ggcactgggt ggcttcttgc 2820 ttgctgtcgt ctacggttcg gctccgtttgtgcccagttc cgccctaggg ttggggctag 2880 ccgcgatcgc ggcctattgg gccctgctctcgctgacaga tatcgatctg cggcaagcaa 2940 cccccattca ctggctggtg ctgctctactggggcgtcga tgccctagca acgggactct 3000 cacccgtacg cgctgcagct ttagttgggctagccaaact gacgctctac ctgttggttt 3060 ttgccctagc ggctcgggtt ctccgcaatccccgtctgcg atcgctgctg ttctcggtcg 3120 tcgtgatcac atcgcttttt gtcagtgtctacggcctcaa ccaatggatc tacggcgttg 3180 aagagctggc gacttgggtg gatcgcaactcggttgccga cttcacctca cgggtttaca 3240 gctatctggg caaccccaac ctgctggctgcttatctggt gccgacgact gccttttctg 3300 cagcagcgat cggggtgtgg cgcggctggctccccaagct gctggcgatc gctgcgacag 3360 gtgcgagcag cttatgtctg atcctcacctacagtcgcgg tggctggctg ggttttgtcg 3420 ccatgatttt tgtctgggcg ttattagggctctactggtt tcaaccccgt ctacccgcac 3480 cctggcgacg ctggctattc ccagtcgtattgggtggact agtcgcggtg ctcttggtgg 3540 cggtgcttgg acttgagccg ttgcgcgtgcgcgtgttgag catctttgtg gggcgtgaag 3600 acagcagcaa caacttccgg atcaatgtctggctggcggt gctgcagatg attcaagatc 3660 ggccttggct gggcatcggc cccggcaataccgcctttaa cctggtttat cccctctatc 3720 aacaggcgcg ctttacggcg ttgagcgcctactccgtccc gctggaagtc gcggttgagg 3780 gcggactact gggcttgacg gccttcgcttggctgctgct ggtcacggcg gtgacggcgg 3840 tgcggcaggt gagccgactg cggcgcgatcgcaatcccca agccttttgg ttgatggcta 3900 gcttggccgg tttggcagga atgctgggtcacggtctgtt tgataccgtg ctctatcgac 3960 cggaagccag tacgctctgg tggctctgtattggagcgat cgcgagtttc tggcagcccc 4020 aaccttccaa gcaactccct ccagaagccgagcattcaga cgaaaaaatg tagcgggctc 4080 cccaacaaat tcctgtgcac ccgactggatccaccaccta aactggatcc caaaggtatc 4140 cggtggatct agggtcataa cgaactccgaccgcgatcgc gtccgcgaac tgaacctcca 4200 tcgcaccgaa gcggagttcg ttagtcgttgaagagccaat gctagagggg gctgccgaag 4260 cagttgggct ggaagcaggc tgcgagaagccacccgcatc caaggcaaag ttcagccgac 4320 cttccgcaaa gactacgatc gccacggcggctctgccagc taagtcagcg ctgggttagt 4380 tgtcatagca gtccgcagac aagttaggacaacttcatag agggactcgc tcagagtcaa 4440 cagccgctgt ccgtgggggt gcgcaatcacccccacaccc acgcactggg ggactcgact 4500 cccccaggcc ccccgcaaca agatttcggataaggggcat cggctgaatc gcgatcgctg 4560 cgggtaaaac tagccggtgt tagccatgggtttgagacta atcggcacgg ggcaaaacgt 4620 cctgatttat ttgctcaatg tgataggttacatcgtcaaa aacaaggccc aagaggtagg 4680 aaaaatcacg accgcccaag tccgagggctttgctgttgg gagcgaccta gggcagacta 4740 gacagagcat tgctgtgagc caaagcgccttcaattgctg gcggctgtgg gtttttcgga 4800 ggttgccaaa tgaaagacct tttcgtcaatgtcctccgct atccccgcta cttcatcacc 4860 ttccagctgg gtatttttta gtcgatctaccagtgggtgc ggccgatggt tcgcaaccca 4920 gtcgcggctt gggcgctgct aggctttggagtttcga 4957 2 1404 DNA Synechococcus sp. 2 atgactgtct ggcaaactctgacttttgcc cattaccaac cccaacagtg gggccacagc 60 agtttcttgc atcggctgtttggcagcctg cgagcttggc gggcctccag ccagctgttg 120 gtttggtctg aggcactgggtggcttcttg cttgctgtcg tctacggttc ggctccgttt 180 gtgcccagtt ccgccctagggttggggcta gccgcgatcg cggcctattg ggccctgctc 240 tcgctgacag atatcgatctgcggcaagca acccccattc actggctggt gctgctctac 300 tggggcgtcg atgccctagcaacgggactc tcacccgtac gcgctgcagc tttagttggg 360 ctagccaaac tgacgctctacctgttggtt tttgccctag cggctcgggt tctccgcaat 420 ccccgtctgc gatcgctgctgttctcggtc gtcgtgatca catcgctttt tgtcagtgtc 480 tacggcctca accaatggatctacggcgtt gaagagctgg cgacttgggt ggatcgcaac 540 tcggttgccg acttcacctcacgggtttac agctatctgg gcaaccccaa cctgctggct 600 gcttatctgg tgccgacgactgccttttct gcagcagcga tcggggtgtg gcgcggctgg 660 ctccccaagc tgctggcgatcgctgcgaca ggtgcgagca gcttatgtct gatcctcacc 720 tacagtcgcg gtggctggctgggttttgtc gccatgattt ttgtctgggc gttattaggg 780 ctctactggt ttcaaccccgtctacccgca ccctggcgac gctggctatt cccagtcgta 840 ttgggtggac tagtcgcggtgctcttggtg gcggtgcttg gacttgagcc gttgcgcgtg 900 cgcgtgttga gcatctttgtggggcgtgaa gacagcagca acaacttccg gatcaatgtc 960 tggctggcgg tgctgcagatgattcaagat cggccttggc tgggcatcgg ccccggcaat 1020 accgccttta acctggtttatcccctctat caacaggcgc gctttacggc gttgagcgcc 1080 tactccgtcc cgctggaagtcgcggttgag ggcggactac tgggcttgac ggccttcgct 1140 tggctgctgc tggtcacggcggtgacggcg gtgcggcagg tgagccgact gcggcgcgat 1200 cgcaatcccc aagccttttggttgatggct agcttggccg gtttggcagg aatgctgggt 1260 cacggtctgt ttgataccgtgctctatcga ccggaagcca gtacgctctg gtggctctgt 1320 attggagcga tcgcgagtttctggcagccc caaccttcca agcaactccc tccagaagcc 1380 gagcattcag acgaaaaaatgtag 1404 3 467 PRT Synechococcus sp. 3 Met Thr Val Trp Gln Thr Leu ThrPhe Ala His Tyr Gln Pro Gln Gln 1 5 10 15 Trp Gly His Ser Ser Phe LeuHis Arg Leu Phe Gly Ser Leu Arg Ala 20 25 30 Trp Arg Ala Ser Ser Gln LeuLeu Val Trp Ser Glu Ala Leu Gly Gly 35 40 45 Phe Leu Leu Ala Val Val TyrGly Ser Ala Pro Phe Val Pro Ser Ser 50 55 60 Ala Leu Gly Leu Gly Leu AlaAla Ile Ala Ala Tyr Trp Ala Leu Leu 65 70 75 80 Ser Leu Thr Asp Ile AspLeu Arg Gln Ala Thr Pro Ile His Trp Leu 85 90 95 Val Leu Leu Tyr Trp GlyVal Asp Ala Leu Ala Thr Gly Leu Ser Pro 100 105 110 Val Arg Ala Ala AlaLeu Val Gly Leu Ala Lys Leu Thr Leu Tyr Leu 115 120 125 Leu Val Phe AlaLeu Ala Ala Arg Val Leu Arg Asn Pro Arg Leu Arg 130 135 140 Ser Leu LeuPhe Ser Val Val Val Ile Thr Ser Leu Phe Val Ser Val 145 150 155 160 TyrGly Leu Asn Gln Trp Ile Tyr Gly Val Glu Glu Leu Ala Thr Trp 165 170 175Val Asp Arg Asn Ser Val Ala Asp Phe Thr Ser Arg Val Tyr Ser Tyr 180 185190 Leu Gly Asn Pro Asn Leu Leu Ala Ala Tyr Leu Val Pro Thr Thr Ala 195200 205 Phe Ser Ala Ala Ala Ile Gly Val Trp Arg Gly Trp Leu Pro Lys Leu210 215 220 Leu Ala Ile Ala Ala Thr Gly Ala Ser Ser Leu Cys Leu Ile LeuThr 225 230 235 240 Tyr Ser Arg Gly Gly Trp Leu Gly Phe Val Ala Met IlePhe Val Trp 245 250 255 Ala Leu Leu Gly Leu Tyr Trp Phe Gln Pro Arg LeuPro Ala Pro Trp 260 265 270 Arg Arg Trp Leu Phe Pro Val Val Leu Gly GlyLeu Val Ala Val Leu 275 280 285 Leu Val Ala Val Leu Gly Leu Glu Pro LeuArg Val Arg Val Leu Ser 290 295 300 Ile Phe Val Gly Arg Glu Asp Ser SerAsn Asn Phe Arg Ile Asn Val 305 310 315 320 Trp Leu Ala Val Leu Gln MetIle Gln Asp Arg Pro Trp Leu Gly Ile 325 330 335 Gly Pro Gly Asn Thr AlaPhe Asn Leu Val Tyr Pro Leu Tyr Gln Gln 340 345 350 Ala Arg Phe Thr AlaLeu Ser Ala Tyr Ser Val Pro Leu Glu Val Ala 355 360 365 Val Glu Gly GlyLeu Leu Gly Leu Thr Ala Phe Ala Trp Leu Leu Leu 370 375 380 Val Thr AlaVal Thr Ala Val Arg Gln Val Ser Arg Leu Arg Arg Asp 385 390 395 400 ArgAsn Pro Gln Ala Phe Trp Leu Met Ala Ser Leu Ala Gly Leu Ala 405 410 415Gly Met Leu Gly His Gly Leu Phe Asp Thr Val Leu Tyr Arg Pro Glu 420 425430 Ala Ser Thr Leu Trp Trp Leu Cys Ile Gly Ala Ile Ala Ser Phe Trp 435440 445 Gln Pro Gln Pro Ser Lys Gln Leu Pro Pro Glu Ala Glu His Ser Asp450 455 460 Glu Lys Met 465 4 1425 DNA Synechococcus sp. 4 atggtgtctcccatctctat ctggcgatcg ctgatgtttg gcggtttttc cccccaggaa 60 tggggccggggcagtgtgct ccatcgtttg gtgggctggg gacagagttg gatacaggct 120 agtgtgctctggccccactt cgaggcattg ggtacggctc tagtggcaat aatttttatt 180 gcggctcccttcacctccac caccatgttg ggcattttta tgctgctctg tggagccttt 240 tgggctctgctgacctttgc tgatcaacca gggaagggtt tgactcccat ccatgtttta 300 gtttttgcctactggtgcat ttcggcgatc gccgtgggat tttctccggt aaaaatggcg 360 gcggcgtcggggttagcgaa attaacagct aatttatgtc tgtttctact ggcggcgagg 420 ttattgcaaaacaaacaatg gttgaaccgg ttagtaaccg ttgttttact ggtagggcta 480 ttggtggggagttacggtct gcgacaacag gtggacgggg tagaacagtt agccacttgg 540 aatgaccccacctctacctt ggcccaggcc actagggtat atagcttttt aggtaatccc 600 aatctcttggcggcttacct ggtgcccatg acgggtttga gcttgagtgc cctggtggta 660 tggcgacggtggtggcccaa actgctggga gcaaccatgg tgattgttaa cctactctgt 720 ctcttttttacccagagccg gggcggttgg ctagcagtgc tggccctggg agctaccttc 780 ctggccctttgttacttctg gtggttaccc caattaccca aattttggca acggtggtct 840 ttgcccctggcgatcgccgt ggcggttata ttaggtgggg gagcgttgat tgcggtggaa 900 ccgattcgactcagggccat gagcattttt gctgggcggg aagacagcag taataatttc 960 cgcatcaatgtttgggaagg ggtaaaagcc atgatccgag cccgccctat cattggcatt 1020 ggcccaggtaacgaagcctt taaccaaatt tatccttact atatgcggcc ccgcttcacc 1080 gccctgagtgcctattccat ttacctagaa attttggtgg aaacgggtgt agttggtttt 1140 acctgtatgctctggctgtt ggccgttacc ctaggcaaag gcgtagaact ggttaaacgc 1200 tgtcgccaaaccctcgcccc ggaaggcatc tggattatgg gggctttagc ggcgatcatc 1260 ggtttgttggtccacggcat ggtagataca gtctggtacc gtcccccggt gagcactttg 1320 tggtggttgctagtggccat tgttgctagt cagtgggcca gcgcccaggc ccgtttggag 1380 gccagtaaagaagaaaatga ggacaaacct cttcttgctt cataa 1425 5 474 PRT Synechococcus sp.5 Met Val Ser Pro Ile Ser Ile Trp Arg Ser Leu Met Phe Gly Gly Phe 1 5 1015 Ser Pro Gln Glu Trp Gly Arg Gly Ser Val Leu His Arg Leu Val Gly 20 2530 Trp Gly Gln Ser Trp Ile Gln Ala Ser Val Leu Trp Pro His Phe Glu 35 4045 Ala Leu Gly Thr Ala Leu Val Ala Ile Ile Phe Ile Ala Ala Pro Phe 50 5560 Thr Ser Thr Thr Met Leu Gly Ile Phe Met Leu Leu Cys Gly Ala Phe 65 7075 80 Trp Ala Leu Leu Thr Phe Ala Asp Gln Pro Gly Lys Gly Leu Thr Pro 8590 95 Ile His Val Leu Val Phe Ala Tyr Trp Cys Ile Ser Ala Ile Ala Val100 105 110 Gly Phe Ser Pro Val Lys Met Ala Ala Ala Ser Gly Leu Ala LysLeu 115 120 125 Thr Ala Asn Leu Cys Leu Phe Leu Leu Ala Ala Arg Leu LeuGln Asn 130 135 140 Lys Gln Trp Leu Asn Arg Leu Val Thr Val Val Leu LeuVal Gly Leu 145 150 155 160 Leu Val Gly Ser Tyr Gly Leu Arg Gln Gln ValAsp Gly Val Glu Gln 165 170 175 Leu Ala Thr Trp Asn Asp Pro Thr Ser ThrLeu Ala Gln Ala Thr Arg 180 185 190 Val Tyr Ser Phe Leu Gly Asn Pro AsnLeu Leu Ala Ala Tyr Leu Val 195 200 205 Pro Met Thr Gly Leu Ser Leu SerAla Leu Val Val Trp Arg Arg Trp 210 215 220 Trp Pro Lys Leu Leu Gly AlaThr Met Val Ile Val Asn Leu Leu Cys 225 230 235 240 Leu Phe Phe Thr GlnSer Arg Gly Gly Trp Leu Ala Val Leu Ala Leu 245 250 255 Gly Ala Thr PheLeu Ala Leu Cys Tyr Phe Trp Trp Leu Pro Gln Leu 260 265 270 Pro Lys PheTrp Gln Arg Trp Ser Leu Pro Leu Ala Ile Ala Val Ala 275 280 285 Val IleLeu Gly Gly Gly Ala Leu Ile Ala Val Glu Pro Ile Arg Leu 290 295 300 ArgAla Met Ser Ile Phe Ala Gly Arg Glu Asp Ser Ser Asn Asn Phe 305 310 315320 Arg Ile Asn Val Trp Glu Gly Val Lys Ala Met Ile Arg Ala Arg Pro 325330 335 Ile Ile Gly Ile Gly Pro Gly Asn Glu Ala Phe Asn Gln Ile Tyr Pro340 345 350 Tyr Tyr Met Arg Pro Arg Phe Thr Ala Leu Ser Ala Tyr Ser IleTyr 355 360 365 Leu Glu Ile Leu Val Glu Thr Gly Val Val Gly Phe Thr CysMet Leu 370 375 380 Trp Leu Leu Ala Val Thr Leu Gly Lys Gly Val Glu LeuVal Lys Arg 385 390 395 400 Cys Arg Gln Thr Leu Ala Pro Glu Gly Ile TrpIle Met Gly Ala Leu 405 410 415 Ala Ala Ile Ile Gly Leu Leu Val His GlyMet Val Asp Thr Val Trp 420 425 430 Tyr Arg Pro Pro Val Ser Thr Leu TrpTrp Leu Leu Val Ala Ile Val 435 440 445 Ala Ser Gln Trp Ala Ser Ala GlnAla Arg Leu Glu Ala Ser Lys Glu 450 455 460 Glu Asn Glu Asp Lys Pro LeuLeu Ala Ser 465 470 6 31 DNA Artificial sequence Syntheticoligonucleotide 6 gggctagccg cgatcgcggc ctattgggcc c 31 7 27 DNAArtificial sequence Synthetic oligonucleotide 7 gggctaggga tcgcgcctattgggccc 27 8 26 DNA Artificial sequence Synthetic oligonucleotide 8gggctcagat cgcgcctatt gggccc 26 9 11 PRT Synechococcus sp. 9 Gly Leu AlaAla Ile Ala Ala Tyr Trp Ala Leu 1 5 10

What is claimed is:
 1. A method of enhancing inorganic carbon fixationby a photosynthetic organism, the method comprising the step oftransforming cells of the photosynthetic organism with an expressiblepolynucleotide encoding a polypeptide having a bicarbonate transporteractivity.
 2. The method of claim 1, wherein said step of transformingsaid cells of the photosynthetic organism with said expressiblepolynucleotide encoding said bicarbonate transporter is effected by amethod selected from the group consisting of genetic transformation andtransient transformation.
 3. The method of claim 2, wherein said genetictransformation is effected by a method selected from the groupconsisting of Agrobaterium mediated transformation, electroporation andparticle bombardment.
 4. The method of claim 2, wherein said transienttransformation is effected by a method selected from the groupconsisting of viral transformation, electroporation and particlebombardment.
 5. The method of claim 1, wherein said expressiblepolynucleotide includes: (i) a nucleic acid sequence corresponding to atleast a portion derived from SEQ ID NO:2, said portion encodes saidprotein having said bicarbonate transporter activity; (ii) a nucleicacid sequence at least 60% identical to said portion, as determinedusing the Blast software where gap penalty equals 10 for existence and10 for extension, average match equals 10 and average mismatch equals−5; (iii) a nucleic acid segment hybridizable with said portion underhybridization conditions of hybridization solution containing 10%dextrane sulfate, 1 M NaCl, 1% SDS and 5×10⁶ cpm ³²p labeled probe, at65° C., with a final wash solution of 0.2× SSC and 0.1% SDS and finalwash at 65° C.; (iv) a man induced variation of said portion; or (v) anaturally occurring variation of said portion.
 6. The method of claim 1,wherein said polypeptide is at least 70% homologous to SEQ ID NO:3 or aportion thereof having said bicarbonate transporter activity asdetermined using the Blast software where gap open penalty equals 11,gap extension penalty equals 1 and matrix is blosum62.
 7. The method ofclaim 1, wherein the photosynthetic organism is a plant.
 8. The methodof claim 7, wherein said plant is a C3 plant.
 9. The method of claim 8,wherein said C3 plant is selected from the group consisting of tobacco,tomato, soybeans, potato, cucumber, cotton, wheat, rice and barley. 10.The method of claim 7, wherein said plant is a C4 plant.
 11. The methodof claim 10, wherein said C4 plant is selected from the group consistingof corn, sugar cane and sorghum.
 12. The method of claim 1, wherein saidpolynucleotide further includes a plant promoter.
 13. The method ofclaim 12, wherein said plant promoter is selected from the groupconsisting of a constitutive plant promoter, a tissue specific plantpromoter and an inducible plant promoter.
 14. The method of claim 13,wherein: (i) said constitutive plant promoter is independently selectedfrom the group consisting of CaMV35 S plant promoter, CaMV19S plantpromoter, FMV34S plant promoter, sugarcane bacilliform badnavirus plantpromoter, CSVMV plant promoter, Arabidopsis ACT2/ACT8 actin plantpromoter, Arabidopsis ubiquitin UBQ1 plant promoter, barley leaf thioninBTH6 plant promoter, and rice actin plant promoter; (ii) said tissuespecific plant promoter is independently selected from the groupconsisting of bean phaseolin storage protein plant promoter, DLEC plantpromoter, PHSβ plant promoter, zein storage protein plant promoter,conglutin gamma plant promoter from soybean, AT2S1 gene plant promoter,ACT11 actin plant promoter from Arabidopsis, napA plant promoter fromBrassica napus and potato patatin gene plant promoter; and (iii) saidinducible plant promoter is independently selected from the groupconsisting of a light-inducible plant promoter derived from the pea rbcSgene, a plant promoter from the alfalfa rbcS gene, DRE, MYC and MYBplant promoters which are active in drought; INT, INPS, prxEa, Hahsp17.7G4 and RD21 plant promoters active in high salinity and osmoticstress, and hsr203J and str246C plant promoters active in pathogenicstress.
 15. The method of claim 1, wherein said polynucleotide furtherincludes a sequence element selected from the group consisting of anucleic acid sequence encoding a transit peptide, an origin ofreplication for propagation in bacterial cells, at least one sequenceelement for integration into a plant's genome, a polyadenylationrecognition sequence, a transcription termination signal, a sequenceencoding a translation start site, a sequence encoding a translationstop site, plant RNA virus derived sequences, plant DNA virus derivedsequences, tumor inducing (Ti) plasmid derived sequences and atransposable element derived sequence.
 16. A nucleic acid molecule forenhancing inorganic carbon fixation by a photosynthetic organism, thenucleic acid molecule comprising a polynucleotide encoding a polypeptidehaving a bicarbonate transporter activity.
 17. The nucleic acid moleculeof claim 16, further comprising a plant promoter being upstream to thepolynucleotide effective in expressing said polypeptide in a plant. 18.The nucleic acid molecule of claim 16, wherein said polynucleotideincludes: (i) a nucleic acid sequence corresponding to at least aportion derived from SEQ ID NO:2, said portion encodes said proteinhaving said bicarbonate transporter activity; (ii) a nucleic acidsequence at least 60% identical to said portion, as determined using theBlast software where gap penalty equals 10 for existence and 10 forextension, average match equals 10 and average mismatch equals −5; (iii)a nucleic acid segment hybridizable with said portion underhybridization conditions of hybridization solution containing 10%dextrane sulfate, 1 M NaCl, 1% SDS and 5×10⁶ cpm ³²p labeled probe, at65° C., with a final wash solution of 0.2× SSC and 0.1% SDS and finalwash at 65° C.; (iv) a man induced variation of said portion; or (v) anaturally occurring variation of said portion.
 19. The nucleic acidmolecule of claim 16, wherein said polypeptide is at least 70%homologous to SEQ ID NO:3 or a portion thereof having said bicarbonatetransporter activity as determined using the Blast software where gapopen penalty equals 11, gap extension penalty equals 1 and matrix isblosum62.
 20. The nucleic acid molecule of claim 17, wherein said plantpromoter is selected from the group consisting of a constitutive plantpromoter, a tissue specific plant promoter and an inducible plantpromoter.
 21. The nucleic acid molecule of claim 20, wherein: (i) saidconstitutive plant promoter is independently selected from the groupconsisting of CaMV35S plant promoter, CaMV19S plant promoter, FMV34Splant promoter, sugarcane bacilliform badnavirus plant promoter, CsVMVplant promoter, Arabidopsis ACT2/ACT8 actin plant promoter, Arabidopsisubiquitin UBQ1 plant promoter, barley leaf thionin BTH6 plant promoter,and rice actin plant promoter; (ii) said tissue specific plant promoteris independently selected from the group consisting of bean phaseolinstorage protein plant promoter, DLEC plant promoter, PHSβ plantpromoter, zein storage protein plant promoter, conglutin gamma plantpromoter from soybean, AT2S1 gene plant promoter, ACT11 actin plantpromoter from Arabidopsis, napA plant promoter from Brassica napus andpotato patatin gene plant promoter; and (iii) said inducible plantpromoter is independently selected from the group consisting of alight-inducible plant promoter derived from the pea rbcS gene, a plantpromoter from the alfalfa rbcS gene, DRE, MYC and MYB plant promoterswhich are active in drought; INT, INPS, prxEa, Ha hsp17.7G4 and RD21plant promoters active in high salinity and osmotic stress, and hsr203Jand str246C plant promoters active in pathogenic stress.
 22. The nucleicacid molecule of claim 16, further comprising a sequence elementselected from the group consisting of an origin of replication forpropagation in bacterial cells, at least one sequence element forintegration into a plant's genome, a polyadenylation recognitionsequence, a transcription termination signal, a sequence encoding atranslation start site, a sequence encoding a translation stop site,plant RNA virus derived sequences, plant DNA virus derived sequences,tumor inducing (Ti) plasmid derived sequences and a transposable elementderived sequence.
 23. A transformed photosynthetic organism comprisingthe nucleic acid molecule of claim
 16. 24. A transformed photosyntheticorganism comprising the nucleic acid molecule of claim
 17. 25. Thetransformed photosynthetic organism of claim 16, wherein thephotosynthetic organism is a plant.
 26. The transformed photosyntheticorganism of claim 25, wherein said plant is a C3 plant.
 27. Thetransformed photosynthetic organism of claim 26, wherein said C3 plantis selected from the group consisting of tobacco, tomato, soybeans,potato, cucumber, cotton, wheat, rice and barley.
 28. The transformedphotosynthetic organism of claim 25, wherein said plant is a C4 plant.29. The transformed photosynthetic organism of claim 28, wherein said C4plant is selected from the group consisting of corn, sugar cane andsorghum.
 30. The transformed photosynthetic organism of claim 23,wherein said organism is characterized by a photosynthetic rate at least10% higher as compared to a control non-transformed organism underotherwise identical conditions.